The invention relates to immunological methods for diagnosis of conditions characterized by abnormal levels of PTEN protein.
PTEN tumor suppressor gene mutations are the most frequent genetic lesion in the highest incidence female genital tract tumor, endometrioid endometrial adenocarcinoma. The role of PTEN in mediating risk conferred by an abnormal hormonal environment and endometrial hyperplasias is uncertain, because of inadequate reagents for protein expression studies in situ and controversy in precancer diagnosis.
Somatic mutation or deletion of the PTEN tumor suppressor gene has been reported in approximately 40% (1;2) and 40 (3)-76% (4), respectively, of endometrial adenocarcinomas. Further evidence for PTEN function within the female reproductive tract is evident in Pten knockout mice that develop complex proliferative endometrial lesions (5). In humans, familial inheritance of mutant PTEN alleles in Cowden syndrome causes multi-organ development of benign hamartomatous and malignant epithelial tumors (6-8), including an elevated incidence of endometrial adenocarcinoma.
A particular variant of endometrial cancer contributes the bulk of PTEN mutations. These are endometrioid endometrial adenocarcinomas (1;2), the most common type [80% (9;10)] of endometrial cancer in the U.S., readily discriminated by routine histopathology from xe2x80x9cnon-endometrioidxe2x80x9d tumors such as papillary serous and clear cell adenocarcinomas, which also occur at this site. Risk for endometrioid endometrial adenocarcinomas increases in subjects with high estrogen levels unopposed by progestins (11), and appearance of a physically distinctive precancerous lesion (12). Interaction between genetic and hormonal events during the premalignant phases of endometrial tumorigenesis is expected, yet has never been precisely elucidated.
Inaccessibility of premalignant tissues, controversy in their diagnosis, and paucity of high-yield candidate genes are longstanding barriers to productive exploration of endometrial precancer biology. PCR methods have improved the analytical repertoire suited to physically small precancers, including detailed mutational (13), clonal (14), and even lineage reconstruction (15) analysis. However, accurate diagnosis of the precancers themselves, typically termed xe2x80x9chyperplasiasxe2x80x9d in the widely used World Health Organization nomenclature (16), has been difficult to standardize (17), and even when criteria are agreed upon, reproducibility (18) is suboptimal. Previous reports of PTEN mutation in putative endometrial precancers have used subjective diagnostic criteria (19-21).
Thus, there presently is a need for objective, reproducible, and sensitive methods for diagnosis of endometrial precancers.
Loss of PTEN function by mutational or other mechanisms is an early and progressive event in endometrial tumorigenesis that may occur in response to known endocrine risk factors, and offers an immunohistochemical biomarker for premalignant disease. Unexpectedly, it has been discovered that individual PTEN-negative glands in endometria (particularly in subjects exposed to unopposed estrogen) are distinguishable by immunohistochemistry using PTEN antibodies or antigen-binding fragments thereof, and that these glands are the earliest recognizable stage of endometrial carcinogenesis, followed by proliferation into dense clusters that form discrete premalignant lesions. Accordingly, the invention provides improved methods and compositions for diagnosing endometrial precancers. The methods of the invention also may be used for identifying individuals at risk for endometrial cancer, or individuals at risk for the recurrence of endometrial cancer after treatment. The methods of the invention may also be used for identifying pharmaceutical candidate compounds active in the onset, progression, or regression of endometrial cancer or precancer.
According to one aspect of the invention, methods for determining the likelihood of a group of endometrial cells or an endometrial gland to become cancerous are provided. The methods include performing PTEN antibody or antigen-binding fragment thereof immunohistochemistry on a group of endometrial cells or one or more endometrial glands and determining the binding of the PTEN antibody or antigen-binding fragment thereof to the group of endometrial cells or glands. A reduced amount of PTEN antibody or antigen-binding fragment thereof bound to the group of endometrial cells or glands relative to a control group of cells indicates that the group of endometrial cells or glands has an increased likelihood of becoming cancerous.
In preferred embodiments, the PTEN antibody or antigen-binding fragment thereof is 6H2.1 antibody or antigen-binding fragment thereof.
In certain embodiments, the group of endometrial cells or the one or more endometrial glands and the control group of cells are present in a tissue sample, such as a tissue biopsy. In other embodiments the control group of cells and the group of endometrial cells or glands are the same cell type. In certain preferred embodiments, the amount of binding of the PTEN antibody or antigen-binding fragment thereof to the group of endometrial cells or glands is 50% or less of the binding of the PTEN antibody or antigen-binding fragment thereof to the control group of cells.
In still other aspects of the invention, the methods also include determining the size of a group of endometrial cells or one or more endometrial glands which have reduced PTEN expression. The size of the cells or glands can be measured in control tissues or in a control biopsy of the same subject to establish a baseline size or average size for the cells or glands. Likewise, PTEN expression can be measured in control tissues or in control biopsies of the same subject. In these embodiments, an increased size of the group of endometrial cells or the glands relative to a control group of cells or glands indicates that the group of endometrial cells or glands has an increased likelihood of becoming cancerous.
In some embodiments of the foregoing methods, the group of endometrial cells or glands are obtained from a subject suspected of having endometrial cancer, or from a subject having or suspected of having elevated unopposed estrogen levels. In still other embodiments, the subject is receiving or has received unopposed estrogen treatment.
According to another aspect of the invention, methods for determining regression, progression or onset of a condition characterized by abnormal levels of PTEN protein are provided. The methods include obtaining a level of the amount of PTEN from a sample obtained from a subject and comparing the level to a control as a determination of regression, progression or onset of the condition. In preferred embodiments of these methods, the PTEN antibody is 6H2.1. In certain embodiments, the subject is undergoing drug therapy for a condition characterized by abnormal levels of PTEN protein.
According to still another aspect of the invention, methods for monitoring the progression of endometrial precancers are provided. The methods include determining the expression of PTEN in endometrial cells or glands by PTEN antibody or antigen-binding fragment thereof immunohistochemistry of an endometrial tissue sample obtained at a first time, determining the expression of PTEN in endometrial cells or glands by PTEN antibody or antigen-binding fragment thereof immunohistochemistry of an endometrial tissue sample obtained at a second time, and comparing the expression of PTEN in the endometrial cells or glands at the first time and the second time. Reduced expression of PTEN at the second time relative to the first time indicates progression of endometrial precancers to a cancerous stage. In preferred embodiments the PTEN antibody is 6H2.1.
In some embodiments, the methods also include determining the size of groups of endometrial cells or glands which have reduced PTEN expression in the endometrial tissue sample obtained at the first time and the endometrial tissue sample obtained at the second time, and comparing the size of the groups of endometrial cells or the glands which have reduced PTEN expression at the first time and the second time. Increased size of the groups of endometrial cells or the glands which have reduced PTEN expression at the second time relative to the first time indicates progression of endometrial precancers to a cancerous stage.
In certain embodiments, the endometrial tissue samples are obtained from a subject suspected of having endometrial cancer or from a subject having or suspected of having elevated unopposed estrogen levels. In other embodiments, the subject is receiving or has received unopposed estrogen treatment or the subject is undergoing drug therapy for endometrial precancer or endometrial cancer.
Similar methods are useful for determining regression of endometrial cancers or precancers, such as following therapeutic invention. Thus, in another aspect of the invention, methods for monitoring the regression of endometrial precancers are provided. The methods include the steps and materials recited above for determination of progression of endometrial precancers. In the analysis of the results, increased expression of PTEN at the second time relative to the first time indicates regression of endometrial precancers from a cancerous stage, and decreased size of the groups of endometrial cells or the glands which have increased PTEN expression at the second time relative to the first time indicates regression of endometrial precancers from a cancerous stage.
In a further aspect of the invention, methods of selecting a treatment for endometrial precancer or endometrial cancer in a subject are provided. The methods include obtaining a level of PTEN expression from an endometrial tissue sample obtained from the subject by immunohistochemical analysis, and selecting the treatment for endometrial precancer or endometrial cancer in the subject based at least in part on the level obtained. These methods also can be used for evaluating a treatment, e.g., to determine its effectiveness.
In certain embodiments, the subject is already receiving drug therapy for endometrial precancer or endometrial cancer. In preferred embodiments, the immunohistochemical analysis is performed using 6H2.1 clone PTEN-reactive monoclonal antibody.
In still another aspect of the invention, kits are provided for identifying endometrial precancer cells in an endometrial tissue sample. The kits include a container containing a PTEN antibody or antigen-binding fragment thereof, a second container containing a detectable compound for detecting the presence of the PTEN antibody or antigen-binding fragment thereof in a sample and instructions for binding the PTEN antibody or antigen-binding fragment thereof to an endometrial tissue sample and for measuring the amount of PTEN antibody or antigen-binding fragment thereof bound to the endometrial tissue sample using the detectable compound. The detectable compounds include, for example, enzyme substrates, detectably labeled second antibodies, etc.
In some embodiments the kits also include a container containing a second detectable compound for histological determination of the size of groups of cells or glands in the endometrial tissue sample.
Other kits for identifying endometrial precancer cells in an endometrial tissue according to the invention include a container containing a PTEN antibody or antigen-binding fragment thereof conjugated to a detectable compound, a second container containing a second detectable compound for histological determination of the size of groups of cells or glands in the endometrial tissue sample and instructions for binding the PTEN antibody or antigen-binding fragment thereof to an endometrial tissue sample and for measuring the amount of PTEN antibody or antigen-binding fragment thereof bound to the endometrial tissue sample using the detectable compound. The detectable compounds include, for example, radiolabels, fluorescent labels, colorimetric compounds.
According to a further aspect of the invention, methods for diagnosing endometrial precancer in a subject are provided. The methods include: obtaining a biological sample of endometrial tissue or cells from a subject, contacting the sample with an endometrial cell marker that specifically binds to endometrial cells, contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds PTEN, determining specific binding between the antibody or antigen-binding fragment thereof and PTEN in the sample, and determining the specific binding between the endometrial cell marker, and agents in the sample. In some embodiments, the determination of specific binding of the antibody or antigen-binding fragment thereof and the specific binding of the endometrial cell marker in the sample, are compared to the specific binding of the antibody or antigen-binding fragment thereof and the specific binding of the endometrial cell marker in a control group of cells as a diagnosis for endometrial precancer in the subject.
In some embodiments, the antibody or antigen-binding fragment thereof is 6H2.1 antibody or an antigen-binding fragment thereof. In some embodiments the endometrial cell marker is selected from the group consisting of: antibodies and antigen-binding fragments thereof, and ligands. In preferred embodiments, the endometrial cell marker comprises an anti-estrogen receptor antibody or an anti-progesterone receptor antibody. More preferably, the anti-estrogen receptor antibody is ER-ID5 and/or the anti-progesterone receptor antibody is IA6. In some embodiments, the endometrial cell marker comprises estrogen or progesterone. In some embodiments, the sample is menstrual fluid.
In some embodiments, the subject is not suspected of having endometrial cancer, and in other embodiments, the subject is not suspected of having endometrial precancer. In some embodiments, the sample is obtained from a subject having or suspecting of having elevated unopposed estrogen levels and in still other embodiments, the sample is obtained from a subject receiving or having received unopposed estrogen treatment.
According to another aspect of the invention, methods for diagnosing endometrial precancer in a subject are provided. The methods include obtaining a biological sample of endometrial tissue or cells from a subject, isolating the endometrial tissue or cells from the sample, contacting the isolated endometrial tissue or cells with an antibody or antigen-binding fragment thereof that specifically binds PTEN, determining specific binding between the antibody or antigen-binding fragment thereof and PTEN in the isolated tissue or cell sample as a diagnosis for endometrial precancer in the subject. Preferred embodiments include comparing the level of specific binding between the antibody or antigen-binding fragment thereof and PTEN in the isolated tissue or cell sample, and the level of specific binding between the antibody or antigen-binding fragment thereof and PTEN in a matched control tissue or cell sample, as a diagnosis for endometrial precancer in the subject.
In preferred embodiments, the antibody or antigen-binding fragment thereof is 6H2.1 antibody or an antigen-binding fragment thereof. In some embodiments, the sample is menstrual fluid. In certain embodiments, the subject is not suspected of having endometrial cancer, and in other embodiments, the subject is not suspected of having endometrial precancer. In some embodiments, the sample is obtained from a subject having or suspecting of having elevated unopposed estrogen levels, and in some embodiments, the sample is obtained from a subject receiving or having received unopposed estrogen treatment.
According to another aspect of the invention kits for diagnosing endometrial precancer in a subject are provided. The kits include antibodies or antigen-binding fragments thereof that specifically bind PTEN, one or more endometrial cell markers, one or more control molecules, and instructions for the use of the antibodies or antigen-binding fragments thereof, cell markers, and control molecules in the diagnosis of endometrial precancer. In some embodiments, the antibodies or antigen-binding fragments thereof are bound to a substrate. In preferred embodiments, the antibody or antigen-binding fragment thereof is 6H2.1 antibody or an antigen-binding fragment thereof. In certain embodiments, the subject is not suspected of having endometrial cancer, and in other embodiments, the subject is not suspected of having endometrial precancer. In some embodiments, the sample is obtained from a subject having or suspecting of having elevated unopposed estrogen levels, and in some embodiments, the sample is obtained from a subject receiving or having received unopposed estrogen treatment.
According to another aspect of the invention, methods for diagnosing endometrial precancer in a subject are provided. The methods include obtaining a biological sample of endometrial tissue or cells from a subject, determining the level of expression of PTEN in the sample, and determining the level of expression of an endometrial cell-associated molecule in the sample. In a preferred embodiment, the method includes comparing the levels of expression of PTEN and one or more endometrial cell-associated molecules to the level of expression of PTEN and one or more endometrial cell-associated molecules in a control sample. In some embodiments, the endometrial cell-associated molecule is selected from the group consisting of: estrogen receptor polypeptides and progesterone receptor polypeptides. In some embodiments, the levels of PTEN and endometrial cell-associated molecule are determined with nucleic acid amplification methods. In some embodiments, the levels of expression of PTEN and the endometrial cell-associated molecules are determined with an immunoassay. In some embodiments, the sample is a body tissue or bodily fluid. In some preferred embodiments, the sample is menstrual fluid. In some preferred embodiments, the sample is endometrial tissue. In certain embodiments, the subject is not suspected of having endometrial cancer, and in other embodiments, the subject is not suspected of having endometrial precancer. In some embodiments, the sample is obtained from a subject having or suspecting of having elevated unopposed estrogen levels, and in some embodiments, the sample is obtained from a subject receiving or having received unopposed estrogen treatment.
According to another aspect of the invention, kits for the diagnosis of endometrial precancer are provided. The kits include oligonucleotides that selectively amplify a nucleic acid sequence that encodes PTEN, oligonucleotides useful for amplifying the nucleic acid sequence that encode one or more endometrial cell-associated molecules, and control nucleic acid primers. In some embodiments the endometrial cell-associated molecule is selected from the group consisting of: estrogen receptor polypeptides or progesterone receptor polypeptides.
According to another aspect of the invention, methods for evaluating the effect of candidate pharmacological compounds on endometrial precancer cell phenotype are provided. The methods include culturing endometrial tissue or cells, contacting the cultured endometrial tissue or cells with an antibody or antigen fragment thereof that specifically binds to PTEN, determining a first amount of specific binding of the antibody or antigen fragment thereof with the endometrial tissue or cells, and contacting the cultured endometrial tissue or cells with a candidate pharmacological agent. The method also includes contacting the cultured endometrial tissue or cells with the antibody or antigen-binding fragment thereof, determining a second amount of specific binding of the antibody or antigen-binding fragment thereof, with the cultured endometrial tissue or cells, and comparing the first and second amounts of specific binding of the antibody or antigen-binding fragment thereof to the tissue or cells. A change in the second amount of specific binding of the antibody or antigen-binding fragment thereof, relative to the first amount of specific binding of the antibody or antigen-binding fragment thereof, indicates the candidate pharmacological compound alters the level of PTEN, wherein a decrease in the relative amount of PTEN indicates the onset of or progression of an endometrial precancer cell phenotype, and where an increase in the relative amount of PTEN indicates the regression of an endometrial precancer cell phenotype. In preferred embodiments, the antibody or antigen-binding fragment thereof is 6H2.1 antibody or an antigen-binding fragment thereof.
In some embodiments, the endometrial tissue or cells are not suspected of having endometrial cancer and in some embodiments, the tissue or cells are not suspected of having endometrial precancer. In some embodiments, the tissue or cells are from a body tissue or bodily fluid, and in some embodiments, the cells are from menstrual fluid. In some embodiments, the tissue or cells are endometrial tissue or cells. In some embodiments, the tissue or cells have not been exposed to unopposed estrogen levels.
According to yet another aspect of the invention methods for evaluating the effect of candidate pharmacological compounds on endometrial precancer cell phenotype are provided. The methods include culturing two matched samples of endometrial tissue or cells, contacting one of the endometrial tissue or cell cultures with a candidate pharmacological agent, contacting each of the endometrial tissue or cell cultures with an antibody or antigen fragment thereof that specifically binds to PTEN, determining the amount of specific binding of the antibody or antigen fragment thereof of each of the endometrial tissue or cell cultures, and comparing the amounts of specific binding of the antibody or antigen-binding fragment thereof to the tissue or cell cultures. A difference in the amount of specific binding of the antibody or antigen-binding fragment thereof in the culture contacted with the candidate pharmacological agent, relative to the amount of specific binding of the antibody or antigen-binding fragment thereof in the culture not contacted with the candidate pharmacological agent, indicates the candidate pharmacological compound alters the level of PTEN. A decrease in the relative amount of PTEN indicates the onset of or progression of an endometrial precancer cell phenotype, and where an increase in the relative amount of PTEN indicates the regression of an endometrial precancer cell phenotype. In preferred embodiments, the antibody or antigen-binding fragment thereof is 6H2.1 antibody or an antigen-binding fragment thereof. In some embodiments, the endometrial tissue or cells are not suspected of having endometrial cancer, and in some embodiments, the tissue or cells are not suspected of having endometrial precancer. In some embodiments, the tissue or cells are from a body tissue or bodily fluid, and in some embodiments the cells are from menstrual fluid. In some embodiments, the tissue or cells are endometrial tissue or cells. In some embodiments, the tissue or cells have not been exposed to unopposed estrogen levels.
The foregoing methods include immunohistochemistry and antibody-based methods but one of ordinary skill in the art would realize that alternative methods for quantifying expression of PTEN would also be useful, including but not limited to: hybridization, selective amplification of nucleic acid molecules encoding PTEN and endometrial cell marker molecules.
The foregoing assay also can be used for assessing exposure to environmental estrogens, such as pesticides.
These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.